1. Field of the Invention
The present invention relates generally to the field of molecular pharmacology. More specifically, the invention involves the molecular biology of the adenylyl cyclase pathway and, in particular, the structure and function of the enzyme adenylyl cyclase.
2. Description of the Related Art
Cyclic AMP regulates intracellular reactions in all nucleated animal cells studied to date. The system by which cyclic AMP is produced is the adenylyl cyclase system, which comprises G-protein coupled receptors, G-proteins and the catalytic, membrane-bound enzyme known as adenylyl cyclase. Adenylyl cyclases have molecular weights of about 120 kD and are stimulated directly by the diterpene forskolin.
The structures of G protein-regulated adenylyl cyclases are complex, consisting of two intensely hydrophobic domains (M.sub.1 and M.sub.2 with each hypothesized to contain six transmembrane helices) and two approximately 40 kD cytosolic domains (C.sub.1 and C.sub.2). C.sub.1 and C.sub.2 contain sequences (C.sub.1a and C.sub.2a) that are similar to each other, to corresponding regions of related adenylyl cyclases and to the catalytic domains of the related membrane-bound, soluble guanylyl cyclases (Tang and Gilman, 1992). Analysis of a series of truncation and alanine-scanning mutants of mammalian adenylyl cyclases indicated that both C.sub.1a and C.sub.2a (but not C.sub.1b and C.sub.2b) are necessary for catalytic activity (Tang et al., 1992).
Unfortunately, membrane-bound adenylyl cyclases are found in small amounts and the enzymes are both labile and difficult to manipulate in detergent-containing solutions. As a result, biochemical studies on the mechanism of regulation of adenylyl cyclases have been relatively unrewarding. Improved methods for isolation of this enzyme, relying on a forskolin affinity matrix, have permitted some purification. Recombinant expression has proved difficult as well, as the significant transmembrane regions of adenylyl cyclases create significant technical problems, especially in prokaryotic expression systems. Thus, there remains a need to develop improved reagents and assays that can be used to further characterize adenylyl cyclase and to screen for compounds that stimulate and inhibit adenylyl cyclase activity.